N-acetyl-l-cysteine protects dental tissue stem cells against oxidative stress in vitro

  • Jasmina Martacic
  • Milica Kovacevic Filipovic
  • Suncica Borozan
  • Zorica Cvetkovic
  • Tamara Popovic
  • Aleksandra Arsic
  • Marija Takic
  • Vesna Vucic
  • Maria Glibetic
Original Article
  • 19 Downloads

Abstract

Objectives

The aim of our study was to investigate whether N-acetyl-l-cysteine (NAC) could protect stem cells from exfoliated deciduous teeth (SHED) against oxidative damage, during in vitro cultivation, to preserve regenerative potential of these cells. Accordingly, we examined the potential of cell culture supplementation with NAC in prevention of lipid peroxidation, unfavorable changes of total lipids fatty acid composition, and the effects on the activity of antioxidant enzymes.

Material and methods

We analyzed the extent of oxidative damage in SHED after 48 h treatment with different NAC concentrations. Cellular lipid peroxidation was determined upon reaction with thiobarbituric acid. All enzyme activities were measured spectrophotometrically, based on published methods. Fatty acid methyl esters were analyzed by gas-liquid chromatography.

Results

Concentration of 0.1 mM NAC showed the most profound effects on SHED, significantly decreasing levels of lipid peroxidation in comparison to control. This dose also diminished the activities of antioxidant enzymes. Furthermore, NAC treatment significantly changed fatty acid composition of cells, reducing levels of oleic acid and monounsaturated fatty acids and increasing linoleic acid, n-6, and total polyunsaturated fatty acid (PUFA) proportions.

Conclusion

Low dose of NAC significantly decreased lipid peroxidation and altered fatty acid composition towards increasing PUFA. The reduced oxidative damage of cellular lipids could be strongly related to improved SHED survival in vitro.

Clinical relevance

Low doses of antioxidants, applied during stem cells culturing and maintenance, could improve cellular characteristics in vitro. This is prerequisite for successful use of stem cells in various clinical applications.

Keywords

N-acetyl-l-cysteine Exfoliated deciduous teeth stem cells Lipid peroxidation Fatty acid composition 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, Geurtsen W (2011) Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth. Calcif Tissue Int 88(2):130–141.  https://doi.org/10.1007/s00223-010-9438-0 CrossRefPubMedGoogle Scholar
  2. 2.
    Karadzic I, Vucic V, Jokanovic V, Debeljak-Martacic J, Markovic D, Petrovic S, Glibetic M (2015) Effects of novel hydroxyapatite-based 3D biomaterials on proliferation and osteoblastic differentiation of mesenchymal stem cells. J Biomed Mater Res Part A 103(1):350–357.  https://doi.org/10.1002/jbm.a.35180 CrossRefGoogle Scholar
  3. 3.
    Xiao Y, Li X, Cui Y, Zhang J, Liu L, Xie X, Hao H, He G, Kander MC, Chen M, Liu Z, Verfaillie CM, Zhu H, Lei M, Liu Z (2014) Hydrogen peroxide inhibits proliferation and endothelial differentiation of bone marrow stem cells partially via reactive oxygen species generation. Life Sci 112(1–2):33–40.  https://doi.org/10.1016/j.lfs.2014.07.016 CrossRefPubMedGoogle Scholar
  4. 4.
    Esmaeli S, Allameh A, Soleimani M, Rahbarizadeh F, Frouzandeh-Moghadam M (2014) The role of albumin and PPAR-α in differentiation-dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte-like cells. Cell Biochem Funct 32(5):410–419.  https://doi.org/10.1002/cbf.3031 PubMedGoogle Scholar
  5. 5.
    Das UN (2006) Essential fatty acids: biochemistry, physiology and pathology. Biotechnol J 1(4):420–439.  https://doi.org/10.1002/biot.200600012 CrossRefPubMedGoogle Scholar
  6. 6.
    Kelly GS (1998) Clinical applications of N-acetylcysteine. Altern Med Rev 3(2):114–127PubMedGoogle Scholar
  7. 7.
    Fan J, Cai H, Yang S, Yan L, Tan W (2008) Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34(+) cells. Comp Biochem Physiol B Biochem Mol Biol 151(2):153–158.  https://doi.org/10.1016/j.cbpb.2008.06.008 CrossRefPubMedGoogle Scholar
  8. 8.
    Song H, Cha MJ, Song BW, Kim IK, Chang W, Lim S, Choi EJ, Ham O, Lee SY, Chung N, Jang Y, Hwang KC (2010) Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex. Stem Cells 28(3):555–563.  https://doi.org/10.1002/stem.302 PubMedGoogle Scholar
  9. 9.
    Martacić JD, Francuski J, Luzajić T, Vuković N, Mojsilović S, Drndarević N, Petakov M, Glibetić M, Marković D, Radovanović A, Todorović V, Filipović MK (2014) Characterization of deciduous teeth stem cells isolated from crown dental pulp. Vojnosanit Pregl 71(8):735–741.  https://doi.org/10.2298/VSP1408735D CrossRefPubMedGoogle Scholar
  10. 10.
    Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, Caplan AI, Cerruti HF (2006) Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs 184(3–4):105–116.  https://doi.org/10.1159/000099617 CrossRefPubMedGoogle Scholar
  11. 11.
    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317.  https://doi.org/10.1080/14653240600855905 CrossRefPubMedGoogle Scholar
  12. 12.
    Cynamon HA, Isenberg JN, Nguyen CH (1984) A rapid method for erythrocyte membrane phospholipid determination. Clin Chim Acta 144(1):65–70.  https://doi.org/10.1016/0009-8981(84)90261-4 CrossRefPubMedGoogle Scholar
  13. 13.
    Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77.  https://doi.org/10.1016/0003-9861(59)90090-6 CrossRefPubMedGoogle Scholar
  14. 14.
    Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478.  https://doi.org/10.1016/0076-6879(90)86141-H CrossRefPubMedGoogle Scholar
  15. 15.
    Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126.  https://doi.org/10.1016/S0076-6879(84)05016-3 CrossRefPubMedGoogle Scholar
  16. 16.
    Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247(10):3170–3175PubMedGoogle Scholar
  17. 17.
    Radovanović T, Borković Mitić S, Perendija B, Despotović S, Pavlović Z, Cakić PD, Saičić Z (2010) Superoxide dismutase and catalase activities in the liver and muscle of barbel (Barbus barbus) and its intestinal parasite (Pomphoryinchus laevis) from the Danube river, Serbia. Arch Biol Sci 62(1):97–105.  https://doi.org/10.2298/ABS1001097R CrossRefGoogle Scholar
  18. 18.
    Rose HG, Oklander M (1965) Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res 6:428–431PubMedGoogle Scholar
  19. 19.
    Ristić-Medić D, Suzić S, Vučić V, Takić M, Tepšić J, Glibetić M (2009) Serum and erythrocyte membrane phospholipids fatty acid composition in hyperlipidemia: effects of dietary intervention and combined diet and fibrate therapy. Gen Physiol Biophys 28:190–199CrossRefPubMedGoogle Scholar
  20. 20.
    Ates B, Abraham L, Ercal N (2008) Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC). Free Radic Res 42(4):372–377.  https://doi.org/10.1080/10715760801998638 CrossRefPubMedGoogle Scholar
  21. 21.
    Samuni Y, Goldstein S, Dean OM, Berk M (2013) The chemistry and biological activities of N-acetylcysteine. Biochim Biophys Acta 1830(8):4117–4129.  https://doi.org/10.1016/j.bbagen.2013.04.016 CrossRefPubMedGoogle Scholar
  22. 22.
    Francik R, Krośniak M, Sanocka I, Bartoń H, Hebda T, Francik S (2014) Aronia melanocarpa treatment and antioxidant status in selected tissues in Wistar rats. Biomed Res Int 2014:457085.  https://doi.org/10.1155/2014/457085 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Debeljak Martacic J, Borozan S, Radovanovic A, Popadic D, Mojsilovic S, Vucic V, Todorovic V, Kovacevic Filipovic M (2016) N-acetyl-l-cysteine enhances ex-vivo amplification of deciduous teeth dental pulp stem cells. Arch Oral Biol 70:32–38.  https://doi.org/10.1016/j.archoralbio.2016.06.002 CrossRefPubMedGoogle Scholar
  24. 24.
    Dalvi SM, Patil VW, Ramraje NN (2012) The roles of glutathione, glutathione peroxidase, glutathione reductase and the carbonyl protein in pulmonary and extra pulmonary tuberculosis. J Clin Diagn Res 6(9):1462–1465.  https://doi.org/10.7860/JCDR/2012/4410.2533 PubMedPubMedCentralGoogle Scholar
  25. 25.
    Todorcević M, Skugor S, Ruyter B (2010) Alterations in oxidative stress status modulate terminal differentiation in Atlantic salmon adipocytes cultivated in media rich in n-3 fatty acids. Comp Biochem Physiol B Biochem Mol Biol 156(4):309–318.  https://doi.org/10.1016/j.cbpb.2010.04.010 CrossRefPubMedGoogle Scholar
  26. 26.
    Kang JX, Wan JB, He C (2014) Concise review: regulation of stem cell proliferation and differentiation by essential fatty acids and their metabolites. Stem Cells 32(5):1092–1098.  https://doi.org/10.1002/stem.1620 CrossRefPubMedGoogle Scholar
  27. 27.
    Russo GL (2009) Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to clinical implications in cardiovascular prevention. Biochem Pharmacol 77(6):937–946.  https://doi.org/10.1016/j.bcp.2008.10.020 CrossRefPubMedGoogle Scholar
  28. 28.
    Kim MH, Kim MO, Kim YH, Kim JS, Han HJ (2009) Linoleic acid induces mouse embryonic stem cell proliferation via Ca2þ/PKC, PI3K/Akt, and MAPKs. Cell Physiol Biochem 23(1-3):53–64.  https://doi.org/10.1159/000204090 CrossRefPubMedGoogle Scholar
  29. 29.
    Lee JH, Tachibana H, Morinaga Y, Fujimura Y, Yamada K (2009) Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids. Life Sci 84(13-14):415–420.  https://doi.org/10.1016/j.lfs.2009.01.004 CrossRefPubMedGoogle Scholar
  30. 30.
    Mohammadzadeh F, Mosayebi G, Montazeri V, Darabi M, Fayezi S, Shaaker M, Rahmati M, Baradaran B, Mehdizadeh A, Darabi M (2014) Fatty acid composition of tissue cultured breast carcinoma and the effect of stearoyl-CoA desaturase 1 inhibition. J Breast Cancer 17(2):136–142.  https://doi.org/10.4048/jbc.2014.17.2.136 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kilpinen L, Tigistu-Sahle F, Oja S, Greco D, Parmar A, Saavalainen P, Nikkilä J, Korhonen M, Lehenkari P, Käkelä R, Laitinen S (2013) Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality. J Lipid Res 54(3):622–635.  https://doi.org/10.1194/jlr.M030650 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jasmina Martacic
    • 1
  • Milica Kovacevic Filipovic
    • 2
  • Suncica Borozan
    • 2
  • Zorica Cvetkovic
    • 3
    • 4
  • Tamara Popovic
    • 1
  • Aleksandra Arsic
    • 1
  • Marija Takic
    • 1
  • Vesna Vucic
    • 1
  • Maria Glibetic
    • 1
  1. 1.Institute for Medical ResearchUniversity of BelgradeBelgradeSerbia
  2. 2.Faculty of Veterinary MedicineUniversity of BelgradeBelgradeSerbia
  3. 3.Department of HematologyClinical Hospital Center ZemunBelgradeSerbia
  4. 4.Faculty of MedicineUniversity of BelgradeBelgradeSerbia

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